Glycan-binding Receptors Research Articles

A human lectin array for characterizing host-pathogen interactions

A human lectin array has been developed to probe the interactions of innate immune receptors with pathogenic and commensal micro-organisms. Following the successful introduction of a lectin array containing all of the cow C-type carbohydrate-recognition domains (CRDs), a human array described here contains the C-type CRDs as well as CRDs from other classes of sugar-binding receptors, including galectins, siglecs, R-type CRDs, ficolins, intelectins and chitinase-like lectins. The array is constructed with CRDs modified with single-site biotin tags, ensuring that the sugar-binding sites in CRDs are displayed on a streptavidin-coated surface in a defined orientation and are accessible to the surfaces of microbes. A common approach used for expression and display of CRDs from all of the different structural categories of glycan-binding receptors allows comparisons across lectin families. In addition to previously documented protocols for binding of fluorescently-labeled bacteria, methods have been developed for detecting unlabeled bacteria bound to the array by counter-staining with DNA-binding dye. Screening has also been undertaken with viral glycoproteins and bacterial and fungal polysaccharides. The array provides an unbiased screen for sugar ligands that interact with receptors and many show binding not anticipated from earlier studies. For example, some of the galectins bind with high affinity to bacterial glycans that lack lactose or N-acetyllactosamine. The results demonstrate the utility of the human lectin array for providing a unique overview of the interactions of multiple classes of glycan-binding proteins in the innate immune system with different types of micro-organisms.

Limited impact of cancer-derived gangliosides on anti-tumor immunity in colorectal cancer.

Aberrant glycosylation is a key mechanism employed by cancer cells to evade immune surveillance, induce angiogenesis and metastasis, among other hallmarks of cancer. Sialic acids, distinctive terminal glycan structures located on glycoproteins or glycolipids, are prominently upregulated across various tumor types, including colorectal cancer (CRC). Sialylated glycans modulate anti-tumor immune responses through their interactions with Siglecs, a family of glycan-binding receptors with specificity for sialic acid-containing glycoconjugates, often resulting in immunosuppression. In this paper, we investigated the immunomodulatory function of ST3Gal5, a sialyltransferase that catalyzes the addition of α2-3 sialic acids to glycosphingolipids, since lower expression of ST3Gal5 is associated with better survival of CRC patients. We employed CRISPR/Cas9 to knock out the ST3Gal5 gene in two murine CRC cell lines MC38 and CT26. Glycomics analysis confirmed the removal of sialic acids on glycolipids, with no discernible impact on glycoprotein sialylation. Although knocking out ST3Gal5 in both cell lines did not affect in vivo tumor growth, we observed enhanced levels of regulatory T cells in CT26 tumors lacking ST3Gal5. Moreover, we demonstrate that the absence of ST3Gal5 affected size and blood vessel density only in MC38 tumors. In summary, we ascertain that sialylation of glycosphingolipids has a limited influence on the anti-tumor immune response in CRC, despite detecting alterations in the tumor microenvironment, possibly due to a shift in ganglioside abundance.

A unique Ca2+-inhibited C-type lectin in shrimp Fenneropenaeuschinensis

C-type lectins (CTLs) are glycan-binding pattern recognition receptors (PRRs) that can bind to carbohydrates on pathogen surfaces, triggering immune responses in shrimp innate immunity. In this study, a unique Ca2+-inhibited CTL named FcLec was identified and characterized in Chinese shrimp Fenneropenaeus chinensis. The full-length cDNA sequence of FcLec was 976 bp (GenBank accession number KU361826), with a 615 bp open reading frame (ORF) encoding 204 amino acids. FcLec possesses a C-type lectin-like domain (CTLD) containing four conserved cysteines (Cys105, Cys174, Cys192, and Cys200) and two sugar-binding site structures (QPD and LNP). The tertiary structure of FcLec deduced revealed three α-helices and eight β-pleated sheets. The mRNA expression levels of FcLec in hemocytes and the hepatopancreas were markedly elevated after stimulation with Vibrio anguillarum and white spot syndrome virus (WSSV). The recombinant FcLec protein exhibited Ca2+-independent hemagglutination and bacterial agglutination, but these activities were observed only in the presence of EDTA to chelate metal ions. These findings suggest that FcLec plays important and functionally distinct roles in the shrimp's innate immune response to bacteria and viruses, enriching the current understanding of the relationship between CTL activity and Ca2+ in invertebrates.

Reduced Expression of CLEC4G in Neurons Is Associated with Alzheimer's Disease.

CLEC4G, a glycan-binding receptor, has previously been demonstrated to inhibit Aβ generation, yet its brain localization and functions in Alzheimer's disease (AD) are not clear. We explored the localization, function, and regulatory network of CLEC4G via experiments and analysis of RNA-seq databases. CLEC4G transcripts and proteins were identified in brain tissues, with the highest expression observed in neurons. Notably, AD was associated with reduced levels of CLEC4G transcripts. Bioinformatic analyses revealed interactions between CLEC4G and relevant genes such as BACE1, NPC1, PILRA, TYROBP, MGAT1, and MGAT3, all displaying a negative correlation trend. We further identified the upstream transcriptional regulators NR2F6 and XRCC4 for CLEC4G and confirmed a decrease in CLEC4G expression in APP/PS1 transgenic mice. This study highlights the role of CLEC4G in protecting against AD progression and the significance of CLEC4G for AD research and management.

CD301 and LSECtin glycan-binding receptors of innate immune cells serve as prognostic markers and potential predictors of immune response in breast cancer subtypes.

Glycosylation is a prominent posttranslational modification, and alterations in glycosylation are a hallmark of cancer. Glycan-binding receptors, primarily expressed on immune cells, play a central role in glycan recognition and immune response. Here, we used the recombinant C-type glycan-binding receptors CD301, Langerin, SRCL, LSECtin, and DC-SIGNR to recognize their ligands on tissue microarrays (TMA) of a large cohort (n=1859) of invasive breast cancer of different histopathological types to systematically determine the relevance of altered glycosylation in breast cancer. Staining frequencies of cancer cells were quantified in an unbiased manner by a computer-based algorithm. CD301 showed the highest overall staining frequency (40%), followed by LSECtin (16%), Langerin (4%) and DC-SIGNR (0.5%). By Kaplan-Meier analyses, we identified LSECtin and CD301 as prognostic markers in different breast cancer subtypes. Positivity for LSECtin was associated with inferior disease-free survival in all cases, particularly in estrogen receptor positive (ER+) breast cancer of higher histological grade. In triple negative breast cancer, positivity for CD301 correlated with a worse prognosis. Based on public RNA single-cell sequencing data of human breast cancer infiltrating immune cells, we found CLEC10A (CD301) and CLEC4G (LSECtin) exclusively expressed in distinct subpopulations, particularly in dendritic cells and macrophages, indicating that specific changes in glycosylation may play a significant role in breast cancer immune response and progression.

The glycoimmune checkpoint receptor Siglec-7 interacts with T-cell ligands and regulates T-cell activation

Siglec-7 (sialic acid-binding immunoglobulin-like lectin 7) is a glycan-binding immune receptor that is emerging as a significant target of interest for cancer immunotherapy. The physiological ligands that bind Siglec-7, however, remain incompletely defined. In this study, we characterized the expression of Siglec-7 ligands on peripheral immune cell subsets and assessed whether Siglec-7 functionally regulates interactions between immune cells. We found that disialyl core 1 O-glycans are the major immune ligands for Siglec-7 and that these ligands are particularly highly expressed on naïve T-cells. Densely glycosylated sialomucins are the primary carriers of these glycans, in particular a glycoform of the cell-surface marker CD43. Biosynthesis of Siglec-7-binding glycans is dynamically controlled on different immune cell subsets through a genetic circuit involving the glycosyltransferase GCNT1. Siglec-7 blockade was found to increase activation of both primary T-cells and antigen-presenting dendritic cells invitro, indicating that Siglec-7 binds T-cell glycans to regulate intraimmune signaling. Finally, we present evidence that Siglec-7 directly activates signaling pathways in T-cells, suggesting a new biological function for this receptor. These studies conclusively demonstrate the existence of a novel Siglec-7-mediated signaling axis that physiologically regulates T-cell activity. Going forward, our findings have significant implications for the design and implementation of therapies targeting immunoregulatory Siglec receptors.

Impact of Asialoglycoprotein Receptor and Mannose Receptor Deficiency on Murine Plasma N-glycome Profiles

The asialoglycoprotein receptor (ASGPR) and the mannose receptor C-type 1 (MRC1) are well known for their selective recognition and clearance of circulating glycoproteins. Terminal galactose and N-Acetylgalactosamine are recognized by ASGPR, while terminal mannose, fucose, and N-Acetylglucosamine are recognized by MRC1. The effects of ASGPR and MRC1 deficiency on the N-glycosylation of individual circulating proteins have been studied. However, the impact on the homeostasis of the major plasma glycoproteins is debated and their glycosylation has not been mapped with high molecular resolution in this context. Therefore, we evaluated the total plasma N-glycome and plasma proteome of ASGR1 and MRC1 deficient mice. ASGPR deficiency resulted in an increase in O-acetylation of sialic acids accompanied by higher levels of apolipoprotein D, haptoglobin, and vitronectin. MRC1 deficiency decreased fucosylation without affecting the abundance of the major circulating glycoproteins. Our findings confirm that concentrations and N-glycosylation of the major plasma proteins are tightly controlled and further suggest that glycan-binding receptors have redundancy, allowing compensation for the loss of one major clearance receptor.

GlycoCAP: A Cell-Free, Bacterial Glycosylation Platform for Building Clickable Azido-Sialoglycoproteins.

Glycan-binding receptors known as lectins represent a class of potential therapeutic targets. Yet, the therapeutic potential of targeting lectins remains largely untapped due in part to limitations in tools for building glycan-based drugs. One group of desirable structures is proteins with noncanonical glycans. Cell-free protein synthesis systems have matured as a promising approach for making glycoproteins that may overcome current limitations and enable new glycoprotein medicines. Yet, this approach has not been applied to the construction of proteins with noncanonical glycans. To address this limitation, we develop a cell-free glycoprotein synthesis platform for building noncanonical glycans and, specifically, clickable azido-sialoglycoproteins (called GlycoCAP). The GlycoCAP platform uses an Escherichia coli-based cell-free protein synthesis system for the site-specific installation of noncanonical glycans onto proteins with a high degree of homogeneity and efficiency. As a model, we construct four noncanonical glycans onto a dust mite allergen (Der p 2): α2,3 C5-azido-sialyllactose, α2,3 C9-azido-sialyllactose, α2,6 C5-azido-sialyllactose, and α2,6 C9-azido-sialyllactose. Through a series of optimizations, we achieve more than 60% sialylation efficiency with a noncanonical azido-sialic acid. We then show that the azide click handle can be conjugated with a model fluorophore using both strain-promoted and copper-catalyzed click chemistry. We anticipate that GlycoCAP will facilitate the development and discovery of glycan-based drugs by granting access to a wider variety of possible noncanonical glycan structures and also provide an approach for functionalizing glycoproteins by click chemistry conjugation.

The melanoma tumor glyco-code impacts human dendritic cells' functionality and dictates clinical outcomes.

Subversion of immunity is a hallmark of cancer development. Dendritic cells (DCs) are strategic immune cells triggering anti-tumor immune responses, but tumor cells exploit their versatility to subvert their functions. Tumor cells harbor unusual glycosylation patterns, which can be sensed through glycan-binding receptors (lectins) expressed by immune cells that are crucial for DCs to shape and orientate antitumor immunity. Yet, the global tumor glyco-code and its impact on immunity has not been explored in melanoma. To decrypt the potential link between aberrant glycosylation patterns and immune evasion in melanoma, we investigated the melanoma tumor glyco-code through the GLYcoPROFILE™ methodology (lectin arrays), and depicted its impact on patients' clinical outcome and DC subsets' functionality. Specific glycan patterns correlated with clinical outcome of melanoma patients, GlcNAc, NeuAc, TF-Ag and Fuc motifs being associated with poor outcome, whereas Man and Glc residues elicited better survival. Strikingly, tumor cells differentially impacting cytokine production by DCs harbored distinct glyco-profiles. GlcNAc exhibited a negative influence on cDC2s, whereas Fuc and Gal displayed inhibitory impacts on cDC1s and pDCs. We further identified potential booster glycans for cDC1s and pDCs. Targeting specific glycans on melanoma tumor cells restored DCs' functionality. The tumor glyco-code was also linked to the nature of the immune infiltrate. This study unveils the impact of melanoma glycan patterns on immunity, and paves the way for innovative therapeutic options. Glycans/lectins interactions arise as promising immune checkpoints to rescue DCs from tumor' hijacking to reshape antitumor immunity and inhibit immunosuppressive circuits triggered by aberrant tumor glycosylation.

Identifying Genetic Regulators of Protein-Glycan Interactions with Genome-Wide CRISPR Screening.

Glycans are carbohydrate molecules appended to proteins and lipids on the surface of all living cells. Glycans play key roles in a wide array of biological processes, and structural changes in cell-surface glycosylation patterns have been connected to pathogenesis of several diseases. In particular, cancer cells frequently upregulate expression of glycans that bind to inhibitory receptors (lectins) on immune cells. These glycosylated antigens systematically inhibit immune activity and protect cancer cells from immune surveillance. Understanding how cancer cells generate these glycan ligands can thus lead to identification of novel druggable targets for therapeutic intervention. However, glycan ligand biosynthesis is subject to extremely complex genetic regulation, making it difficult to identify the key genes involved in production of immune-regulatory glycan antigens. In a recent publication, we described a CRISPR/Cas9 screening approach to identify genes that drive synthesis of ligands for glycan-binding immune receptors. Here, we outline a detailed, step-by-step protocol for completing this type of genome-wide screen. Our protocol produces a genome-wide atlas of all genes whose expression is required for cell-surface binding of a recombinant immune lectin. This dataset can be used both to identify novel ligands for immune lectins and annotate regulatory genes that drive changes in cancer-associated glycosylation. Our protocol serves as a general resource for researchers interested in the detailed study of cancer glyco-immunology. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Generation of a genome-wide CRISPR library using lentiviral transduction Support Protocol: Generation of dCas9KRAB-expressing K-562 cells Basic Protocol 2: Staining of genome-wide CRISPR libraries with Siglec-Fc reagents and fluorescence-activated cell sorting Basic Protocol 3: Library amplification and sequencing Basic Protocol 4: Data analysis and hit identification.

Unique CLR expression patterns on circulating and tumor-infiltrating DC subsets correlated with clinical outcome in melanoma patients.

Subversion of immunity by tumors is a crucial step for their development. Dendritic cells (DCs) are strategic immune cells that orchestrate anti-tumor immune responses but display altered functions in cancer. The bases for such DCs' hijacking are not fully understood. Tumor cells harbor unusual glycosylation patterns of surface glycoproteins and glycolipids. DCs express glycan-binding receptors, named C-type lectin receptors (CLR), allowing them to sense changes in glycan signature of their environment, and subsequently trigger a response. Recognition of tumor glycans by CLRs is crucial for DCs to shape antitumor immunity, and decisive in the orientation of the response. Yet the status of the CLR machinery on DCs in cancer, especially melanoma, remained largely unknown. We explored CLR expression patterns on circulating and tumor-infiltrating cDC1s, cDC2s, and pDCs of melanoma patients, assessed their clinical relevance, and further depicted the correlations between CLR expression profiles and DCs' features. For the first time, we highlighted that the CLR repertoire of circulating and tumor-infiltrating cDC1s, cDC2s, and pDCs was strongly perturbed in melanoma patients, with modulation of DCIR, CLEC-12α and NKp44 on circulating DCs, and perturbation of Dectin-1, CD206, DEC205, DC-SIGN and CLEC-9α on tumor-infiltrating DCs. Furthermore, melanoma tumor cells directly altered CLR expression profiles of healthy DC subsets, and this was associated with specific glycan patterns (Man, Fuc, GlcNAc) that may interact with DCs through CLR molecules. Notably, specific CLR expression profiles on DC subsets correlated with unique DCs' activation status and functionality and were associated with clinical outcome of melanoma patients. Higher proportions of DCIR-, DEC205-, CLEC-12α-expressing cDCs were linked with a better survival, whereas elevated proportions of CD206-, Dectin1-expressing cDCs and NKp44-expressing pDCs were associated with a poor outcome. Thus, melanoma tumor may shape DCs' features by exploiting the plasticity of the CLR machinery. Our study revealed that melanoma manipulates CLR pathways to hijack DC subsets and escape from immune control. It further paved the way to exploit glycan-lectin interactions for the design of innovative therapeutic strategies, which exploit DCs' potentialities while avoiding hijacking by tumor, to properly reshape anti-tumor immunity by manipulating the CLR machinery.

Probing the glycans accessibility in the nanoparticle biomolecular corona

HypothesisFollowing blood administration, the pristine surface of nanoparticles (NPs) associates with biomolecules from the surrounding environment forming the so-called “biomolecular corona”. It is well accepted that the biomolecular corona dramatically affects the NP fate in the biological medium while the pristine surface is no longer available for binding. Recent studies have shown that the glycans associated with the proteins forming the corona have a role in the NP interaction with macrophages, but the glycan identities remain unknown. We aim here to identify the glycan composition of the biomolecular corona and to assess the role of these glycans in the interaction of the proteins from the corona with glycan binding biomolecules, such as lectins. ExperimentsIn this study, we have characterized the biomolecular corona of citrate stabilised gold NPs after exposure of the NPs to blood plasma at two different plasma concentrations, mimicking the in vitro and in vivo conditions. We have extensively characterized the biomolecular corona using HILIC chromatography and shotgun proteomics. Following this, a lectin binding assay was carried out using Dynamic Light Scattering (DLS) and Fluorescence Correlation Spectroscopy (FCS) to assess whether proteins with known affinity towards specific glycans would bind to the corona. FindingsOur findings highlighted that the protein corona composition is dependent on the exposing conditions. However, under both plasma concentrations, the biantennary sialylated glycans (A2G2S2) are enriched. DLS and FCS confirmed that the glycans are accessible for binding as the corona interacts with lectins with known affinity towards terminal sialic acids and the enzymatic removal of the glycans leads to a decrease in lectin affinity. This study shows for the first time that the glycans are present in the corona and that they could potentially be responsible for the modulation of NP biological processes as they can directly engage with glycan binding receptors that are highly expressed in an organism.

Siglec-8 Signals Through a Non-Canonical Pathway to Cause Human Eosinophil Death In Vitro.

Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is a glycan-binding receptor bearing immunoreceptor tyrosine-based inhibitory and switch motifs (ITIM and ITSM, respectively) that is selectively expressed on eosinophils, mast cells, and, to a lesser extent, basophils. Previous work has shown that engagement of Siglec-8 on IL-5–primed eosinophils causes cell death via CD11b/CD18 integrin–mediated adhesion and NADPH oxidase activity and identified signaling molecules linking adhesion, reactive oxygen species (ROS) production, and cell death. However, the proximal signaling cascade activated directly by Siglec-8 engagement has remained elusive. Most members of the Siglec family possess similar cytoplasmic signaling motifs and recruit the protein tyrosine phosphatases SHP-1/2, consistent with ITIM-mediated signaling, to dampen cellular activation. However, the dependence of Siglec-8 function in eosinophils on these phosphatases has not been studied. Using Siglec-8 antibody engagement and pharmacological inhibition in conjunction with assays to measure cell-surface upregulation and conformational activation of CD11b integrin, ROS production, and cell death, we sought to identify molecules involved in Siglec-8 signaling and determine the stage of the process in which each molecule plays a role. We demonstrate here that the enzymatic activities of Src family kinases (SFKs), Syk, SHIP1, PAK1, MEK1, ERK1/2, PLC, PKC, acid sphingomyelinase/ceramidase, and Btk are all necessary for Siglec-8–induced eosinophil cell death, with no apparent role for SHP-1/2, SHIP2, or c-Raf. While most of these signaling molecules are necessary for Siglec-8–induced upregulation of CD11b integrin at the eosinophil cell surface, Btk is phosphorylated and activated later in the signaling cascade and is instead necessary for CD11b activation. In contrast, SFKs and ERK1/2 are phosphorylated far earlier in the process, consistent with their role in augmenting cell-surface levels of CD11b. In addition, pretreatment of eosinophils with latrunculin B or jasplakinolide revealed that actin filament disassembly is necessary and sufficient for surface CD11b integrin upregulation and that actin polymerization is necessary for downstream ROS production. These results show that Siglec-8 signals through an unanticipated set of signaling molecules in IL-5–primed eosinophils to induce cell death and challenges the expectation that ITIM-bearing Siglecs signal through inhibitory pathways involving protein tyrosine phosphatases to achieve their downstream functions.

Quantitative Phosphoproteomic Analysis Reveals Dendritic Cell- Specific STAT Signaling After α2-3-Linked Sialic Acid Ligand Binding.

Dendritic cells (DCs) are key initiators of the adaptive immunity, and upon recognition of pathogens are able to skew T cell differentiation to elicit appropriate responses. DCs possess this extraordinary capacity to discern external signals using receptors that recognize pathogen-associated molecular patterns. These can be glycan-binding receptors that recognize carbohydrate structures on pathogens or pathogen-associated patterns that additionally bind receptors, such as Toll-like receptors (TLRs). This study explores the early signaling events in DCs upon binding of α2-3 sialic acid (α2-3sia) that are recognized by Immune inhibitory Sialic acid binding immunoglobulin type lectins. α2-3sias are commonly found on bacteria, e.g. Group B Streptococcus, but can also be expressed by tumor cells. We investigated whether α2-3sia conjugated to a dendrimeric core alters DC signaling properties. Through phosphoproteomic analysis, we found differential signaling profiles in DCs after α2-3sia binding alone or in combination with LPS/TLR4 co-stimulation. α2-3sia was able to modulate the TLR4 signaling cascade, resulting in 109 altered phosphoproteins. These phosphoproteins were annotated to seven biological processes, including the regulation of the IL-12 cytokine pathway. Secretion of IL-10, the inhibitory regulator of IL-12 production, by DCs was found upregulated after overnight stimulation with the α2-3sia dendrimer. Analysis of kinase activity revealed altered signatures in the JAK-STAT signaling pathway. PhosphoSTAT3 (Ser727) and phosphoSTAT5A (Ser780), involved in the regulation of the IL-12 pathway, were both downregulated. Flow cytometric quantification indeed revealed de- phosphorylation over time upon stimulation with α2-3sia, but no α2-6sia. Inhibition of both STAT3 and -5A in moDCs resulted in a similar cytokine secretion profile as α-3sia triggered DCs. Conclusively, this study revealed a specific alteration of the JAK-STAT pathway in DCs upon simultaneous α2-3sia and LPS stimulation, altering the IL10:IL-12 cytokine secretion profile associated with reduction of inflammation. Targeted control of the STAT phosphorylation status is therefore an interesting lead for the abrogation of immune escape that bacteria or tumors impose on the host.

Genome-wide CRISPR screens reveal a specific ligand for the glycan-binding immune checkpoint receptor Siglec-7

Glyco-immune checkpoint receptors, molecules that inhibit immune cell activity following binding to glycosylated cell-surface antigens, are emerging as attractive targets for cancer immunotherapy. Defining biologically relevant ligands that bind and activate such receptors, however, has historically been a significant challenge. Here, we present a CRISPRi genomic screening strategy that allowed unbiased identification of the key genes required for cell-surface presentation of glycan ligands on leukemia cells that bind the glyco-immune checkpoint receptors Siglec-7 and Siglec-9. This approach revealed a selective interaction between Siglec-7 and the mucin-type glycoprotein CD43. Further work identified a specific N-terminal glycopeptide region of CD43 containing clusters of disialylated O-glycan tetrasaccharides that form specific Siglec-7 binding motifs. Knockout or blockade of CD43 in leukemia cells relieves Siglec-7-mediated inhibition of immune killing activity. This work identifies a potential target for immune checkpoint blockade therapy and represents a generalizable approach to dissection of glycan-receptor interactions in living cells.

C-Type Lectins in Veterinary Species: Recent Advancements and Applications.

C-type lectins (CTLs), a superfamily of glycan-binding receptors, play a pivotal role in the host defense against pathogens and the maintenance of immune homeostasis of higher animals and humans. CTLs in innate immunity serve as pattern recognition receptors and often bind to glycan structures in damage- and pathogen-associated molecular patterns. While CTLs are found throughout the whole animal kingdom, their ligand specificities and downstream signaling have mainly been studied in humans and in model organisms such as mice. In this review, recent advancements in CTL research in veterinary species as well as potential applications of CTL targeting in veterinary medicine are outlined.

AB0123 CHANGES IN CELLULAR GLYCOSYLATION AS A KEY FACTOR IN THE IMMUNOPATHOGENESIS OF SYSTEMIC LUPUS ERYTHEMATOSUS

Background:Systemic Lupus Erythematous (SLE) is one of the most challenging autoimmune diseases as it may be presented as a severe, relapsing and disabling immune-mediated disorder still remaining incurable (1,2).Protein glycosylation is an essential post translational modification that participate in the correct recognition of cells by the immune system (3–5). Moreover, glycosylation changes in T cells (specifically loss of branchedN-glycans mediated by GnTV, encoded byMGAT5gene) have been shown to impact its intrinsic function and activity in a diverse panel of autoimmune diseases (3,4,6)Objectives:To evaluate the impact of glycans in the cellular and molecular mechanisms underlying the loss of immune-tolerance, envisioning the identification of a new targeted-specific mechanism.Methods:We have analysed the profile of cellular glycosylation of a subset of biopsy-proven lupus nephritis from SLE patients and normal kidney tissue (from two Porto Centre Hospitals), through immunohistochemistry (IHC) as well as by real time PCR using RNA extracted from paraffin tissues. Blood samples were collected and were analysed by flow cytometry.Mgat5null mice with 15-months old were monitored for autoimmune signs by evaluating proteinuria, weight loss and colon and kidney tissues were analysed by IHC and FACS.Results:SLE patients revealed a significant decreased expression of complexN-glycans in the renal parenchyma, when compared to healthy kidney. In addition, we have identified in lupus nephritis patients a unique subset of circulatory CD3+T cells with an abnormal glycosignature and displaying an increased expression of specific glycan-binding receptors. Interestingly,Mgat5null mice develop clinical signs compatible within autoimmune-like syndrome together with an increased infiltration of specific CD3+T cells subset identified in SLE patients.Conclusion:These findings point towards the identification of a novel immune player with increased ability to sense abnormalN-glycans, modulating the surrounding immune response. We propose glycosylation as a regulatory mechanism that tips the balance between homeostasis/self-tolerance and autoimmunity opening a potential novel targeted-specific mechanism in SLE pathogenesis.

Mammalian lectin arrays for screening host–microbe interactions

Many members of the C-type lectin family of glycan-binding receptors have been ascribed roles in the recognition of microorganisms and serve as key receptors in the innate immune response to pathogens. Other mammalian receptors have become targets through which pathogens enter target cells. These receptor roles have often been documented with binding studies involving individual pairs of receptors and microorganisms. To provide a systematic overview of interactions between microbes and the large complement of C-type lectins, here we developed a lectin array and suitable protocols for labeling of microbes that could be used to probe this array. The array contains C-type lectins from cow, chosen as a model organism of agricultural interest for which the relevant pathogen–receptor interactions have not been previously investigated in detail. Screening with yeast cells and various strains of both Gram-positive and -negative bacteria revealed distinct binding patterns, which in some cases could be explained by binding to lipopolysaccharides or capsular polysaccharides, but in other cases they suggested the presence of novel glycan targets on many of the microorganisms. These results are consistent with interactions previously ascribed to the receptors, but they also highlight binding to additional sugar targets that have not previously been recognized. Our findings indicate that mammalian lectin arrays represent unique discovery tools for identifying both novel ligands and new receptor functions.

Glioblastomas exploit truncated O-linked glycans for local and distant immune modulation via the macrophage galactose-type lectin

Glioblastoma is the most aggressive brain malignancy, for which immunotherapy has failed to prolong survival. Glioblastoma-associated immune infiltrates are dominated by tumor-associated macrophages and microglia (TAMs), which are key mediators of immune suppression and resistance to immunotherapy. We and others demonstrated aberrant expression of glycans in different cancer types. These tumor-associated glycans trigger inhibitory signaling in TAMs through glycan-binding receptors. We investigated the glioblastoma glycocalyx as a tumor-intrinsic immune suppressor. We detected increased expression of both tumor-associated truncated O-linked glycans and their receptor, macrophage galactose-type lectin (MGL), on CD163+ TAMs in glioblastoma patient-derived tumor tissues. In an immunocompetent orthotopic glioma mouse model overexpressing truncated O-linked glycans (MGL ligands), high-dimensional mass cytometry revealed a wide heterogeneity of infiltrating myeloid cells with increased infiltration of PD-L1+ TAMs as well as distant alterations in the bone marrow (BM). Our results demonstrate that glioblastomas exploit cell surface O-linked glycans for local and distant immune modulation.

Synthesis and preliminary evaluation of neoglycopolymers carrying multivalent N-glycopeptide units

In the present study, for the discovery of uncharacterized glycan-binding receptors or lectin-like receptors in plants, we developed neoglycopolymers to which three types of N-glycopeptides are conjugated; the first with plant complex type N-glycan (M3FX), the second with high-mannose type N-glycan (M8), and the third with animal complex type N-glycan (NeuAc2Gal2GN2M3). Three types of Asn-oligosaccharide (Asn-M3FX, Asn-M8, or Asn-NeuAc2Gal2GN2M3) were prepared from storage glycoproteins of Ginkgo biloba seeds, Vigna angularis seeds, and egg yolk glycopeptides from actinase digests of each glycoproteins or glycopeptide. Neoglycopolymers were synthesized such that the α-amino groups of Asn-oligosaccharide were coupled to the carboxyl groups of poly-γ-L-glutamic acid (γ-L-PGA) with 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM). The resulting neoglycopolymers were purified through a combination of gel-filtration and reverse-phase HPLC. The incorporation of N-glycans into γ-L-PGA (mol%) was estimated through amino acid composition analysis after acid hydrolysis. The incorporation rates of Asn-M3FX, Asn-M8, and Asn-NeuAc2Gal2GN2M3 into γ-L-PGA were 15.4%, 8.6%, and 11.1%, indicating that nearly 890, 500, and 640 molecules of N-glycans were conjugated with γ-L-PGA, respectively. Furthermore, we confirmed that the neoglycopolymer carrying the multivalent high-mannose type N-glycans is a useful tool for rapid purification of mannose-binding protein, Concanavalin A, from jack bean extract.